A chemoselective ligation route to glycosyltransferase substrate mimetics

Lead Research Organisation: Queen's University Belfast
Department Name: Sch of Chemistry and Chemical Eng

Abstract

A detailed understanding of the innate workings of the cell is imperative for our prospects of making new advances in the treatment of infections and disease. How the cells of our body respond to stresses ranging from cancer to the common cold is all highly sought knowledge. To help reveal this information we intend to design a range of small chemical compounds which can get into the cells and shut down the function(s) of specific proteins in order to observe the effects on the cell and reveal the role of specific proteins in cell maintenance. This can be achieved by using chemicals which mimic the ones the proteins normally work with and use them to distract the protein from doing its proper job. We want to mimic sugar nucleotides (sugar-NDPs).Sugar-NDPs are the building blocks used by a class of intracellular biocatalytic proteins know as glycosyltransferases (GTs). GTs biosynthesise oligosaccharides and glycoproteins which are central players in cellular function and maintenance. Sugar-NDPs carry some 'fat-repelling' negative charge which stops them from escaping the cells' watery innards through the greasy cell membrane, but also prevents the delivery of sugar-NDP mimics into the cell. The ability to prepare uncharged sugar-NDP mimetics which can ultimately diffuse into cells and perturb the function of specific GTs would be extremely desirable. We will develop synthetic chemical methods that will facilitate the preparation of such sugar-NDP-like compounds by replacing the negatively charged component of the sugar-NDP (ie. the pyrophosphate group) with a sugar molecule. The sugar should be able to mimic the missing pyrophosphate component and also help to make the sugar-NDP mimic more greasy. This will increase its chances of getting into get into the cell across the greasy cell membrane to do the job at hand. Current methods for synthesising such molecules can be particularly challenging and time consuming. However we will design a selection of simple chemical building blocks that be chemically 'clipped' together in different combinations so that it will be possible to prepare a range of uncharged sugar-NDP-like structures in a less labour intensive manner.

Publications

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Iqbal A (2013) Stability of aminooxy glycosides to glycosidase catalysed hydrolysis. in Carbohydrate research

 
Description A central feature of this project was to design small sugar-based chemical compounds which can get into the cells and shut down the function(s) of specific proteins in order to observe the effects on the cell and reveal the role of specific proteins in cell maintenance.



We addressed this by designing simple carbohydrate building blocks that can be chemically 'clipped' together under very mild conditions in a process known as chemoselective ligation.



Key achievements are as follows:-



[1] The pH-dependence of these ligation processes has been established. Under mildly acidic conditions (pH 6) these ligation reactions are very slowly (several days), but give high yields. Reaction under more acidic reaction conditions (e.g. pH 2) are much faster (30 mins), but give low yields. By starting the reaction at pH 2 and slowly increasing to pH 6 over 2 hours we have developed a method enabling high product yields over a short timescale.



[2] We have demonstrated the stability of the unique carbohydrate linkages (formed in these ligation processes) to degradation by cellular hydrolytic enzymes. An important pre-requisite if such molecules are to be used to study processes within cells.



[3] Using these ligation processes we have prepared a small number of non polar (i.e. cell permeable) carbohydrate-based molecules as potential mimics of sugar nucleotides (very polar molecules used in oligosaccharide biosynthesis by enzymes know as glycosyltransferases). These now need to be tested as glycosyltransferase inhibitors both in free solutions and within living cells.



[4] In biological systems, many sugar molecules are chemical bound to specific amino acids within specific proteins (known as glycoproteins). To further extend the utility of this ligation process we have developed some novel and very efficient methods that allow large scale synthesis of special amino acid building blocks (know as keto amino acids) which (once incorporated into synthetic peptides/proteins) it will be possible to selectively glycosylate such elaborate biomolecules at the specific position(s) where the keto-amino acids have been incorporated.



[5] An unexpected (and probably the most significant) discovery from this project was when it became apparent that this type of chemoselective ligation reaction was reversible under reaction conditions which could be tolerated by biological proteins (i.e. mildly acidic aqueous solutions). This has allowed us to establish the viability of this reaction for use in Dynamic combinatorial Chemistry (DCC). DCC involves the formation of reversible chemical bonds between two sets of complementary building block (e.g. A & A' with B and B'), which will give an equilibrium mixture of all the possible reaction products (i.e. AB, AB', A'B and A'B') . When a molecular trap is added (e.g. a protein), if any of the reaction products favorably bind to the protein then the concentrations of these favorably bound molecules is amplified as the dynamic mixture of compound "re-shuffles" to provide more of the favorably binding product. In this manner it is possible to identify potential protein binding ligands (i.e. inhibitors) from within complex mixtures in a manner which is much less laborious than preparing a large number of potential protein-binding inhibitors and testing them individually. We have demonstrate that this chemoselective sugar ligation can be used in such a manner to identify inhibitors of sugar hydrolysing enzymes (called glycosidases) from dynamic compound mixtures, just by mixing a number of appropriate chemical building blocks with the target protein in aqueous solutions buffered at pH 5.



As a result of this work, our next objective will be to further optimise this sugar ligation DCC process and explore it application in the search for carbohydrate tagged molecules as inhibitors of glycosidases in fungal pathogens of high value crops such as tomatoes and potatoes.